Stabilized compositions of proteins having a free thiol moiety

ABSTRACT

Compositions of proteins having free thiols, and methods of making and using such compositions, are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 12/902,680 filed on Oct.12, 2010 which is a continuation of U.S. application Ser. No. 11/671,588filed on Feb. 6, 2007, now U.S. Pat. No. 7,833,766 issued Nov. 16, 2010,which claims priority to U.S. Application Ser. No. 60/771,555, filed onFeb. 7, 2006. The disclosure of the prior application is considered partof (and is incorporated by reference in) the disclosure of thisapplication.

FIELD OF THE INVENTION

The invention relates to compositions of proteins having free thiols,and to methods of making and methods of using such compositions. Thecompositions have optimized stability.

BACKGROUND OF THE INVENTION

A drug product (e.g., that contains a protein) can be stored in liquidor lyophilized, i.e., freeze-dried, form. A lyophilized drug product isoften reconstituted by adding a suitable administration diluent justprior to patient use.

Active protein may be lost as a result of physical instabilities,including denaturation and aggregation, as well as chemicalinstabilities, including, for example, hydrolysis, deamidation, andoxidation. The stability of a protein drug in a particular form, e.g.,in a liquid or in a lyophilized form, can be an important considerationin selection of a product form.

SUMMARY OF THE INVENTION

In general, the invention features a composition which includes aprotein having a free thiol (—S—H) (e.g., on a cysteine residue) and/orother moiety subject to oxidation (e.g., Tyr, Trp, or Met moiety) and acarbohydrate, wherein the carbohydrate is present in an amountsufficient to maintain the stability of the protein, and thereby of thecomposition. In a particularly preferred embodiment, the moiety to beprotected is a free thiol.

Compositions and methods described herein provide for increasedstability and storage life by increasing the stability of a proteincontained therein.

Compositions described herein, e.g., liquid compositions containing aprotein, have prolonged stability. E.g., under pre-selected conditions,e.g., upon storage in a gas tight container, at a temperature of 2-8° C.for a period of up to 3, 6, 9, 12, or 24 months (or in some embodimentslonger), a protein in the composition will retain at least 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 99, or 100% of the stability it had prior tostorage. Stability, as used herein, includes parameters such as proteinstructure (e g, minimizing or preventing changes in protein structure,e.g., protein aggregation or protein degradation (e.g., fragmentation))and/or a biological activity of the protein, e.g., the ability toconvert substrate into product.

Protein stability can be measured, e.g., by measuring proteinaggregation, protein degradation, or levels of a biological activity ofthe protein. Protein aggregation can be determined, e.g., by sizeexclusion chromatography, non-denaturing PAGE, or other methods fordetermining size, etc. For example, the composition can have less than a1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% increase in the amount ofprotein aggregation (e.g., as measured by size exclusion chromatography)as compared to the amount of protein aggregation that was in thecomposition prior to storage (e.g., storage at a temperature of 2-8° C.for a period of up to 3, 6, 9, 12, or 24 months (or longer)). Proteindegradation can be determined, e.g., by reverse phase HPLC,non-denaturing PAGE, ion-exchange chromatography, peptide mapping, orsimilar methods. As an example, the composition can have less than a 1,5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% increase in the amount ofprotein degradation (e.g., as measured by reverse phase HPLC) ascompared to the amount of protein degradation that was in thecomposition prior to storage (e.g., storage at a temperature of 2-8° C.for a period of up to 3, 6, 9, 12, or 24 months (or longer)). Thebiological activity of a protein can be measured, e.g., by in vitro orin vivo assays, e.g., ELISA (e.g., to measure binding or enzymaticactivity) and other enzymatic assays (e.g., spectrophotometric,fluorimetric, calorimetric, chemiluminescent, radiometric, orchromatographic assays), kinase assays, and so forth. As an example, thecomposition can have less than a 1, 5, 10, 15, 20, 25, 30, 35, 40, 45,or 50% decrease in a biological activity of the protein (e.g., enzymaticactivity, e.g., as measured by an in vitro assay) as compared to theamount of the biological activity that was in the composition prior tostorage (e.g., storage at a temperature of 2-8° C. for a period of up to3, 6, 9, 12, or 24 months (or longer)).

In one aspect, the protein does not modify, e.g., cleave, any othercomponents of the composition. For example, in one preferred embodiment,in a composition containing glucocerebrosidase (GCB), the compositiondoes not contain polysorbate as a surfactant because GCB can recognizepolysorbate as a substrate and can cleave polysorbate to release freefatty acids.

Embodiments of the invention have stability comparable to that of alyophilized composition of the same protein. A liquid compositiondescribed herein can have at least 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 99, or 100% of the level of protein stability (e.g., retainedactivity) of a lyophilized composition after 3, 6, 12, 18, or 24 monthsof storage (e.g., if a lyophilized composition has retained 90% of itsactivity at 18 months, the composition of the invention has retained atleast 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% of thatlevel).

In one aspect, the disclosure features a composition that includes aprotein having a free thiol and a carbohydrate, wherein the carbohydrateis present in an amount sufficient to maintain the stability of theprotein and wherein the pH of the composition is less than 7.0. In someembodiments, the composition also includes an antioxidant, wherein theantioxidant and carbohydrate are present in amounts sufficient tomaintain the stability of the protein, and thereby of the composition,and wherein the pH of the composition is less than 7.0. For example, theantioxidant is cysteine, cysteine hydrochloride (cysteine-HCl), ormethionine (e.g., present at between about 0.001 and about 10% (wt/vol))and the carbohydrate is sucrose or trehalose (e.g., present at betweenabout 1 and about 40% (wt/vol)). In certain embodiments, the pH is inthe range of about 4.5 to about 6.5, e.g., preferably between about 5.0and 6.0, e.g., more preferably between about 5.5 and 5.8 (e.g., about5.7). In a preferred embodiment, the composition includes a surfactant(e.g., poloxamer 188).

In a preferred embodiment, the pH of the composition is, e.g., betweenabout 4.5 and about 6.5, e.g., between about 5.0 and about 6.0, e.g.,between about 5.5 and about 5.8 (e.g., about 5.7).

In certain embodiments, the stability is at least 5-80% greater (e.g.,at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, or at least about 80% greater), under pre-selectedconditions, than the stability of a composition which differs by lackingthe carbohydrate (and the antioxidant, if used).

In certain embodiments, the carbohydrate (and optionally, anantioxidant) is present in an amount sufficient to stabilize the freethiol of the protein (e.g., the protein shows less aggregate formation,e.g., the protein shows about 5%, about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90% about 95% or about 99% less aggregate formation under pre-selectedconditions than an otherwise identical protein composition that does notcontain the carbohydrate (and antioxidant, if used)).

In certain embodiments, the carbohydrate (and optionally, anantioxidant) is present in an amount sufficient to increase thestability of the protein (e.g., the protein shows less aggregateformation, e.g., the protein shows about 5%, about 10%, about 15%, about20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90% about 95% or about 99% less aggregate formation underpre-selected conditions than an otherwise identical protein compositionthat does not contain the carbohydrate (and antioxidant, if used)).

In certain embodiments, the carbohydrate (and optionally, anantioxidant) is present in an amount sufficient to inhibit the reactionof a free thiol on a first molecule of the protein with a free thiol ona second molecule of the protein to form an aggregate.

In certain embodiments, the carbohydrate (and optionally, anantioxidant) is present in an amount sufficient to inhibit the formationof an aggregate formed by the reaction of a free thiol on a firstmolecule of the protein with a free thiol on a second molecule of theprotein by at least 5-80% (e.g., at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, or at least about 80%greater), under pre-selected conditions, as compared to the samecomposition lacking the carbohydrate (and the antioxidant, if present).

In certain embodiments, the carbohydrate (and optionally, anantioxidant) is present in an amount sufficient that upon storage, in agas tight container, at a temperature of 2-8° C., for a period of 6months, the composition will retain at least 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 99, or 100% of the stability the composition had prior tostorage. In a preferred embodiment, the storage occurs in darkness.

In certain embodiments, the carbohydrate (and optionally, anantioxidant) is present in an amount sufficient to have stabilitycomparable to that of a lyophilized composition comprising about 0.01%polysorbate-20, pH 6.0, 50 mM Citrate. In certain embodiments, thecomposition further includes about 1-40% (e.g., about 5 to about 30%,e.g., about 8 to about 24%, e.g., about 16%, e.g., about 3-5% weight pervolume (w/v)) of a carbohydrate, e.g., sucrose or trehalose. In someembodiments, the carbohydrate is preferably sucrose.

In a preferred embodiment, the composition is a liquid.

In certain embodiments, the composition contains less than about 10% O₂(e.g., less than about 5% O₂, e.g., less than about 2% O₂). In apreferred embodiment, the amount of dissolved O₂ is less than the amountof dissolved inert gases in the composition.

In certain embodiments, the composition is made by a method comprisingphysical removal of O₂ from the composition (e.g., degassing thecomposition, purging a solution with a gas other than O₂, e.g., with aninert gas (e.g., with N₂ or Ar), e.g., bubbling the gas other than O₂(e.g., N₂ or Ar) through the composition).

In certain embodiments, the protein in the composition that contains afree thiol has zero, two, four, six, or more thiol groups which formsulfhydryl bridges. In certain embodiments, the protein containing afree thiol has two, three, or more free thiol groups and has zero, two,four, or more thiol groups which form sulfhydryl bridges, per activeunit of protein.

In certain embodiments, the protein containing a free thiol is selectedfrom the group consisting of glucocerebrosidase (GCB), basic fibroblastgrowth factor (bFGF), acidic fibroblast growth factor (aFGF),hemoglobin, thioredoxin, calcium- and integrin-binding protein 1 (CIB1),beta-lactoglobulin B, beta-lactoglobulin AB, serum albumin, antibodies(e.g., human antibodies, e.g., IgA (e.g., dimeric IgA), IgG (e.g.,IgG2), and IgM; recombinant human antibodies), antibody fragments (e.g.,Fab′ fragments, F(ab′)₂ fragments, single-chain Fv fragments (scFv)),antibodies and antibody fragments (e.g., Fab′, e.g., monoclonal antibodyfragment C46.3; and scFv) engineered (e.g., so that the antibody orantibody fragment can be labeled, e.g., with 99mTc, to clinical imaging)to introduce cysteine residues (e.g., in the third heavy chain constantdomain, e.g., at position 442 in EU/OU numbering; monoclonal antibodyMN-14 (a high-affinity anti-carcinoembryonic antigen (CEA)mab)), core 2beta 1,6-N-acetylglucosaminyltransferase-M (C2GnT-M), core 2 beta1,6-N-acetylglucosaminyltransferase-I (C2GnT-I), platelet-derived growthfactor receptor-beta (PDGF-beta), adenine nucleotide translocase (ANT),p53 tumor suppressor protein, gluten proteins, acid sphingomyelinase(recombinant acid sphyngomyelinase), desfuroylceftiofur (DFC),apolipoprotein B100 (apoB) and other low density lipoprotein domains,apolipoprotein A-I variants (e.g., apolipoprotein A-I (Milano) andapolipoprotein A-I (Paris)), hypoxia-inducible factor-1 alpha (HIF-1alpha), von Willebrand factor (VWF), proteins and peptide mimetics thatcontain the CAAX motif (e.g., Ras), mucolytics, carboxypeptidase Y,cathepsin B, cathepsin C, skeletal muscle Ca²⁺ release channel/ryanodinereceptor (RyR1), nuclear factor kappa B (NF-KB), AP-1, protein-disulfideisomerase (PDI), glycoprotein 1b alpha (GP1b alpha), calcineurin (CaN),fibrillin-1, CD4, S100A3 (also known as S100E), ionotropic glutamatereceptors, human inter-alpha-inhibitor heavy chain 1, alpha2-antiplasmin(alpha2AP), thrombospondin (also known as glycoprotein G), gelsolin,mucins, creatine kinase (e.g., S-thiomethyl-modified creatine kinase),Factor VIII, phospholipase D (PLD), insulin receptor beta subunit,acetylcholinesterase, prochymosin, modified alpha 2-macroglobulin (alpha2M) (e.g., proteinase- or methylamine-reacted alpha 2M), glutathionereductase (GR), complement component C2 (e.g., 2a), complement componentC3 (e.g., C3b), complement component 4 (e.g., 4d), complement Factor B(e.g., Bb), alpha-lactalbumin, beta-D-galactosidase, endoplasmicreticulum Ca²⁺-ATPase, RNase inhibitor, lipocortin 1 (also known asannexin 1), proliferating cell nuclear antigen (PCNA), actin (e.g.,globular actin), coenzyme A (CoA), acyl-CoA synthetase (e.g.,butyryl-coenzyme A synthetase), 3-2trans-enoyl-CoA-isomerase precursor,atrial natriuretic factor (ANF)-sensitive guanylate cyclase,Pz-peptidase, aldehyde dehydrogenase (e.g., acylated aldehydedehydrogenase), P-450 and NADPH-P-450 reductase,glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 6-pyruvoyltetrahydropterin synthetase, lutropin receptor, low moleculat weightacid phosphatase, serum cholinesterase (BChE), adrenodoxin,hyaluronidase, carnitine acyltransferases, interleukin-2 (IL-2),phosphoglycerate kinase, insulin-degrading enzyme (IDE), cytochrome c1heme subunit, S-protein, valyl-tRNA synthetase (VRS), alpha-amylase I,muscle AMP deaminase, lactate dehydrogenase, and somatostatin-bindingprotein.

In a preferred embodiment, the protein containing a free thiol is GCB.

In another preferred embodiment, the protein containing a free thiol isbFGF.

In one aspect, the disclosure features a liquid composition of GCB thatincludes GCB, and a carbohydrate at a pH less than 7.0, was produced byexposing the composition to an inert gas (e.g., N₂), and the inert gasis present in a concentration higher than in the ambient atmosphere,e.g., the composition contains at least about 85%, 90%, 95%, or 99%, orpreferably 100% inert gas. In certain embodiments, the composition alsoincludes an antioxidant. For example, the antioxidant is cysteine,cysteine-HCl, or methionine (e.g., present at between about 0.001 andabout 10% (wt/vol)) and the carbohydrate is sucrose or trehalose (e.g.,present at between about 1 and about 40% (wt/vol)). In certainembodiments, the pH is in the range of about 4.5 to about 6.5, e.g.,preferably between about 5.0 and about 6.0, e.g., more preferablybetween about 5.5 and about 5.8 (e.g., about 5.7). In certainembodiments, the composition also contains a surfactant (e.g., poloxymer188).

In one aspect, the disclosure features a composition that includes aprotein having a free thiol and a carbohydrate, and is at a pH below thepKa of a free thiol on the protein, wherein the carbohydrate is presentin an amount sufficient to increase the stability of the protein at thepH.

In certain embodiments, the stability is at least 5-80% greater (e.g.,at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, or at least about 80% greater), under pre-selectedconditions, than the stability of a composition which lacks thecarbohydrate and which has a pH above the pKa of a free thiol on theprotein.

In certain embodiments, the carbohydrate is present in an amountsufficient to stabilize the free thiol of the protein.

In certain embodiments, the carbohydrate is present in an amountsufficient to inhibit the reaction of a free thiol on a first moleculeof the protein with a free thiol on a second molecule of the protein toform an aggregate.

In certain embodiments, the carbohydrate is present in an amountsufficient to inhibit the formation of an aggregate formed by thereaction of a free thiol on a first molecule of the protein with a freethiol on a second molecule of the protein by at least 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 99, or 100%, under pre-selected conditions, ascompared to the same composition lacking the carbohydrate.

In a preferred embodiment, the carbohydrate is present in an amountsufficient that upon storage in darkness, in a gas tight container, at atemperature of 2-8° C. for a period of up to 3, 6, 9, 12, or 24 months(or longer), the composition will retain at least 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 99, or 100% of the stability it had prior tostorage.

In certain embodiments, the carbohydrate is present in an amountsufficient for the composition to have stability comparable to that of alyophilized composition.

In a preferred embodiment, the composition is a liquid.

In certain embodiments, the composition contains less than 10% O₂ (e.g.,less than 5% O₂, e.g., less than 2% O₂). In certain embodiments, theamount of dissolved O₂ is less than the amount of dissolved inert gasesin the composition.

In certain embodiments, the composition is made by a method comprisingphysical removal of O₂ from the composition (e.g., degassing thecomposition, purging a solution with a gas other than O₂, e.g., with aninert gas (e.g., with N₂ or Ar), e.g., bubbling the gas other than O₂(e.g., N₂ or Ar) through the composition).

In certain embodiments, the protein in the composition that contains afree thiol has two, three, four, five, or more free thiol groups peractive unit of protein.

In certain embodiments, the protein in the composition that contains afree thiol has two, four, six, or more thiol groups which formsulfhydryl bridges per active unit (e.g., dimer) of protein. In certainembodiments, the protein that contains a free thiol has two, three, ormore free thiol groups and has two, four, or more thiol groups whichform sulfhydryl bridges, per active unit of protein.

In certain embodiments, the protein containing a free thiol is selectedfrom the group consisting of glucocerebrosidase (GCB), basic fibroblastgrowth factor (bFGF), acidic fibroblast growth factor (aFGF),hemoglobin, thioredoxin, calcium- and integrin-binding protein 1 (CIB1),beta-lactoglobulin B, beta-lactoglobulin AB, serum albumin, antibodies(e.g., human antibodies, e.g., IgA (e.g., dimeric IgA), IgG (e.g.,IgG2), and IgM; recombinant human antibodies), antibody fragments (e.g.,Fab′ fragments, F(ab′)₂ fragments, single-chain Fv fragments (scFv)),antibodies and antibody fragments (e.g., Fab′, e.g., monoclonal antibodyfragment C46.3; and scFv) engineered (e.g., so that the antibody orantibody fragment can be labeled, e.g., with 99mTc, to clinical imaging)to introduce cysteine residues (e.g., in the third heavy chain constantdomain, e.g., at position 442 in EU/OU numbering; monoclonal antibodyMN-14 (a high-affinity anti-carcinoembryonic antigen (CEA)mab)), core 2beta 1,6-N-acetylglucosaminyltransferase-M (C2GnT-M), core 2 beta1,6-N-acetylglucosaminyltransferase-I (C2GnT-I), platelet-derived growthfactor receptor-beta (PDGF-beta), adenine nucleotide translocase (ANT),p53 tumor suppressor protein, gluten proteins, acid sphingomyelinase(recombinant acid sphyngomyelinase), desfuroylceftiofur (DFC),apolipoprotein B100 (apoB) and other low density lipoprotein domains,apolipoprotein A-I variants (e.g., apolipoprotein A-I (Milano) andapolipoprotein A-I (Paris)), hypoxia-inducible factor-1 alpha (HIF-1alpha), von Willebrand factor (VWF), proteins and peptide mimetics thatcontain the CAAX motif (e.g., Ras), mucolytics, carboxypeptidase Y,cathepsin B, cathepsin C, skeletal muscle Ca²⁺ release channel/ryanodinereceptor (RyR1), nuclear factor kappa B (NF-KB), AP-1, protein-disulfideisomerase (PDI), glycoprotein 1b alpha (GP1b alpha), calcineurin (CaN),fibrillin-1, CD4, S100A3 (also known as S100E), ionotropic glutamatereceptors, human inter-alpha-inhibitor heavy chain 1, alpha2-antiplasmin(alpha2AP), thrombospondin (also known as glycoprotein G), gelsolin,mucins, creatine kinase (e.g., S-thiomethyl-modified creatine kinase),Factor VIII, phospholipase D (PLD), insulin receptor beta subunit,acetylcholinesterase, prochymosin, modified alpha 2-macroglobulin (alpha2M) (e.g., proteinase- or methylamine-reacted alpha 2M), glutathionereductase (GR), complement component C2 (e.g., 2a), complement componentC3 (e.g., C3b), complement component 4 (e.g., 4d), complement Factor B(e.g., Bb), alpha-lactalbumin, beta-D-galactosidase, endoplasmicreticulum Ca²⁺-ATPase, RNase inhibitor, lipocortin 1 (also known asannexin 1), proliferating cell nuclear antigen (PCNA), actin (e.g.,globular actin), coenzyme A (CoA), acyl-CoA synthetase (e.g.,butyryl-coenzyme A synthetase), 3-2trans-enoyl-CoA-isomerase precursor,atrial natriuretic factor (ANF)-sensitive guanylate cyclase,Pz-peptidase, aldehyde dehydrogenase (e.g., acylated aldehydedehydrogenase), P-450 and NADPH-P-450 reductase,glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 6-pyruvoyltetrahydropterin synthetase, lutropin receptor, low moleculat weightacid phosphatase, serum cholinesterase (BChE), adrenodoxin,hyaluronidase, carnitine acyltransferases, interleukin-2 (IL-2),phosphoglycerate kinase, insulin-degrading enzyme (IDE), cytochrome c1heme subunit, S-protein, valyl-tRNA synthetase (VRS), alpha-amylase I,muscle AMP deaminase, lactate dehydrogenase, and somatostatin-bindingprotein.

In a preferred embodiment, the protein containing a free thiol is GCB.

In another preferred embodiment, the protein containing a free thiol isbFGF.

In one aspect, the disclosure features a liquid composition of GCB thatincludes GCB and a carbohydrate and is at a pH between about 0 and about7, and the carbohydrate is present in an amount sufficient to maintainthe biophysical/biochemical integrity (e.g., molecular weight, chargedistribution) and bioactivity characteristics/properties of the GCB atthe pH. For example, the composition retains at least 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 99, or 100% of the biological activity it hadprior to storage (e.g., storage at a temperature of 2-8° C. for a periodof up to 3, 6, 9, 12, or 24 months (or longer)). As another example, atleast 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% of theproteins in the composition retain the average molecular weight oraverage charge distribution that the proteins had prior to storage(e.g., storage at a temperature of 2-8° C. for a period of up to 3, 6,9, 12, or 24 months (or longer)).

In certain embodiments, the pH is in the range of about 4.5 to about6.5, e.g., about 5.0 to about 6.0 (e.g., the pH is about 5.5 to about5.8, e.g., about 5.7).

In a preferred embodiment, the carbohydrate is sucrose or trehalose(e.g., present in an amount between about 1 and about 40%, e.g., betweenabout 3% and about 5% (wt/vol)).

In one aspect, the disclosure features a liquid composition of GCB thatincludes GCB, an antioxidant, a carbohydrate, at a pH between 4.5-6.5,and the composition was produced by exposing the composition to an inertgas (e.g., N₂ or Ar). In certain embodiments, the pH is in the range ofabout 4.5 to about 6.5, e.g., about 5.0 to about 6.0 (e.g., the pH isabout 5.5 to about 5.8, e.g., about 5.7).

In certain embodiments, the liquid composition includes about 0.1-40mg/ml GCB (e.g., more preferably about 0.5 to about 10 mg/ml, e.g.,about 2 to about 8 mg/ml or about 5 mg/ml) (e.g., about 2 mg/ml), about0.001-10% cysteine (e.g., about 0.075%), about 1-40% sucrose (e.g.,about 16%), at a pH of about 5.5-6.0 (e.g., about 5.7), and the level ofdissolved O₂ is less than about 10% (e.g., less than about 5%, e.g.,less than about 2%).

In a preferred embodiment, the composition also includes a surfactant(e.g., poloxamer 188).

In one aspect, the disclosure features a gas tight container thatcontains a protein component and a headspace wherein the proteincomponent is a protein having a free thiol and the headspace is at least90%, 95% or 99% (vol/vol) an inert gas.

In certain embodiments, the gas tight container is a prefilled syringe,a vial, or an ampoule. In a more preferred embodiment, the prefilledsyringe is a needleless syringe.

In certain embodiments, the protein containing a free thiol is selectedfrom the group consisting of glucocerebrosidase (GCB), basic fibroblastgrowth factor (bFGF), acidic fibroblast growth factor (aFGF),hemoglobin, thioredoxin, calcium- and integrin-binding protein 1 (CIB1),beta-lactoglobulin B, beta-lactoglobulin AB, serum albumin, antibodies(e.g., human antibodies, e.g., IgA (e.g., dimeric IgA), IgG (e.g.,IgG2), and IgM; recombinant human antibodies), antibody fragments (e.g.,Fab′ fragments, F(ab′)₂ fragments, single-chain Fv fragments (scFv)),antibodies and antibody fragments (e.g., Fab′, e.g., monoclonal antibodyfragment C46.3; and scFv) engineered (e.g., so that the antibody orantibody fragment can be labeled, e.g., with 99mTc, to clinical imaging)to introduce cysteine residues (e.g., in the third heavy chain constantdomain, e.g., at position 442 in EU/OU numbering; monoclonal antibodyMN-14 (a high-affinity anti-carcinoembryonic antigen (CEA)mab)), core 2beta 1,6-N-acetylglucosaminyltransferase-M (C2GnT-M), core 2 beta1,6-N-acetylglucosaminyltransferase-I (C2GnT-I), platelet-derived growthfactor receptor-beta (PDGF-beta), adenine nucleotide translocase (ANT),p53 tumor suppressor protein, gluten proteins, acid sphingomyelinase(recombinant acid sphyngomyelinase), desfuroylceftiofur (DFC),apolipoprotein B100 (apoB) and other low density lipoprotein domains,apolipoprotein A-I variants (e.g., apolipoprotein A-I (Milano) andapolipoprotein A-I (Paris)), hypoxia-inducible factor-1 alpha (HIF-1alpha), von Willebrand factor (VWF), proteins and peptide mimetics thatcontain the CAAX motif (e.g., Ras), mucolytics, carboxypeptidase Y,cathepsin B, cathepsin C, skeletal muscle Ca²⁺ release channel/ryanodinereceptor (RyR1), nuclear factor kappa B (NF-KB), AP-1, protein-disulfideisomerase (PDI), glycoprotein 1b alpha (GP1b alpha), calcineurin (CaN),fibrillin-1, CD4, S100A3 (also known as S100E), ionotropic glutamatereceptors, human inter-alpha-inhibitor heavy chain 1, alpha2-antiplasmin(alpha2AP), thrombospondin (also known as glycoprotein G), gelsolin,mucins, creatine kinase (e.g., S-thiomethyl-modified creatine kinase),Factor VIII, phospholipase D (PLD), insulin receptor beta subunit,acetylcholinesterase, prochymosin, modified alpha 2-macroglobulin (alpha2M) (e.g., proteinase- or methylamine-reacted alpha 2M), glutathionereductase (GR), complement component C2 (e.g., 2a), complement componentC3 (e.g., C3b), complement component 4 (e.g., 4d), complement Factor B(e.g., Bb), alpha-lactalbumin, beta-D-galactosidase, endoplasmicreticulum Ca²⁺-ATPase, RNase inhibitor, lipocortin 1 (also known asannexin 1), proliferating cell nuclear antigen (PCNA), actin (e.g.,globular actin), coenzyme A (CoA), acyl-CoA synthetase (e.g.,butyryl-coenzyme A synthetase), 3-2trans-enoyl-CoA-isomerase precursor,atrial natriuretic factor (ANF)-sensitive guanylate cyclase,Pz-peptidase, aldehyde dehydrogenase (e.g., acylated aldehydedehydrogenase), P-450 and NADPH-P-450 reductase,glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 6-pyruvoyltetrahydropterin synthetase, lutropin receptor, low moleculat weightacid phosphatase, serum cholinesterase (BChE), adrenodoxin,hyaluronidase, carnitine acyltransferases, interleukin-2 (IL-2),phosphoglycerate kinase, insulin-degrading enzyme (IDE), cytochrome c1heme subunit, S-protein, valyl-tRNA synthetase (VRS), alpha-amylase I,muscle AMP deaminase, lactate dehydrogenase, and somatostatin-bindingprotein.

In a preferred embodiment, the protein containing a free thiol is GCB.

In another preferred embodiment, the protein containing a free thiol isbFGF.

In one aspect, the disclosure features a method of packaging acomposition that includes contacting a free thiol containing proteinwith an inert gas (e.g., N₂ or Ar) to reduce the amount of a reactivespecies (e.g., O₂), and introducing the protein and the inert gas into agas tight container. The term “reactive species” includes molecules orions formed by the incomplete one-electron reduction of oxygen. Thesereactive species include O₂; superoxides; peroxides; hydroxyl radical;and hypochlorous acid.

In a preferred embodiment, the inert gas is N₂ or Ar and the reactivespecies is O₂.

In certain embodiments, the free thiol containing protein is selectedfrom the group consisting of glucocerebrosidase (GCB), basic fibroblastgrowth factor (bFGF), acidic fibroblast growth factor (aFGF),hemoglobin, thioredoxin, calcium- and integrin-binding protein 1 (CIB1),beta-lactoglobulin B, beta-lactoglobulin AB, serum albumin, antibodies(e.g., human antibodies, e.g., IgA (e.g., dimeric IgA), IgG (e.g.,IgG2), and IgM; recombinant human antibodies), antibody fragments (e.g.,Fab′ fragments, F(ab′)₂ fragments, single-chain Fv fragments (scFv)),antibodies and antibody fragments (e.g., Fab′, e.g., monoclonal antibodyfragment C46.3; and scFv) engineered (e.g., so that the antibody orantibody fragment can be labeled, e.g., with 99mTc, to clinical imaging)to introduce cysteine residues (e.g., in the third heavy chain constantdomain, e.g., at position 442 in EU/OU numbering; monoclonal antibodyMN-14 (a high-affinity anti-carcinoembryonic antigen (CEA)mab)), core 2beta 1,6-N-acetylglucosaminyltransferase-M (C2GnT-M), core 2 beta1,6-N-acetylglucosaminyltransferase-I (C2GnT-I), platelet-derived growthfactor receptor-beta (PDGF-beta), adenine nucleotide translocase (ANT),p53 tumor suppressor protein, gluten proteins, acid sphingomyelinase(recombinant acid sphyngomyelinase), desfuroylceftiofur (DFC),apolipoprotein B100 (apoB) and other low density lipoprotein domains,apolipoprotein A-I variants (e.g., apolipoprotein A-I (Milano) andapolipoprotein A-I (Paris)), hypoxia-inducible factor-1 alpha (HIF-1alpha), von Willebrand factor (VWF), proteins and peptide mimetics thatcontain the CAAX motif (e.g., Ras), mucolytics, carboxypeptidase Y,cathepsin B, cathepsin C, skeletal muscle Ca²⁺ release channel/ryanodinereceptor (RyR1), nuclear factor kappa B (NF-KB), AP-1, protein-disulfideisomerase (PDI), glycoprotein 1b alpha (GP1b alpha), calcineurin (CaN),fibrillin-1, CD4, S100A3 (also known as S100E), ionotropic glutamatereceptors, human inter-alpha-inhibitor heavy chain 1, alpha2-antiplasmin(alpha2AP), thrombospondin (also known as glycoprotein G), gelsolin,mucins, creatine kinase (e.g., S-thiomethyl-modified creatine kinase),Factor VIII, phospholipase D (PLD), insulin receptor beta subunit,acetylcholinesterase, prochymosin, modified alpha 2-macroglobulin (alpha2M) (e.g., proteinase- or methylamine-reacted alpha 2M), glutathionereductase (GR), complement component C2 (e.g., 2a), complement componentC3 (e.g., C3b), complement component 4 (e.g., 4d), complement Factor B(e.g., Bb), alpha-lactalbumin, beta-D-galactosidase, endoplasmicreticulum Ca²⁺-ATPase, RNase inhibitor, lipocortin 1 (also known asannexin 1), proliferating cell nuclear antigen (PCNA), actin (e.g.,globular actin), coenzyme A (CoA), acyl-CoA synthetase (e.g.,butyryl-coenzyme A synthetase), 3-2trans-enoyl-CoA-isomerase precursor,atrial natriuretic factor (ANF)-sensitive guanylate cyclase,Pz-peptidase, aldehyde dehydrogenase (e.g., acylated aldehydedehydrogenase), P-450 and NADPH-P-450 reductase,glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 6-pyruvoyltetrahydropterin synthetase, lutropin receptor, low moleculat weightacid phosphatase, serum cholinesterase (BChE), adrenodoxin,hyaluronidase, carnitine acyltransferases, interleukin-2 (IL-2),phosphoglycerate kinase, insulin-degrading enzyme (IDE), cytochrome c1heme subunit, S-protein, valyl-tRNA synthetase (VRS), alpha-amylase I,muscle AMP deaminase, lactate dehydrogenase, and somatostatin-bindingprotein.

In a preferred embodiment, the protein containing a free thiol is GCB.

In another preferred embodiment, the protein containing a free thiol isbFGF.

In one aspect, the disclosure features a method of treating a patient(e.g., a patient in need of treatment with a free-thiol containingprotein, e.g., a patient with a deficiency of the free-thiol protein)that includes administering a composition described herein, e.g., acomposition containing a free-thiol protein (e.g., GCB), to a patient.For example, a pharmaceutical composition that is administered to apatient can include a composition described herein, e.g., in atherapeutically-effective amount.

In a preferred embodiment, the administration is by IV infusion orsubcutaneous.

In one embodiment, a composition described herein that contains afree-thiol protein (e.g., GCB) is used in therapy.

In one embodiment, a composition described herein that contains afree-thiol protein (e.g., GCB) is used for the manufacture of amedicament for the treatment of a condition in which there is a need forthe free-thiol containing protein (e.g., the use of a GCB compositiondescribed herein for the treatment of a glucocerebrosidase deficiency,e.g., Gaucher disease). For example, a medicament for administration toa patient can include a composition described herein, e.g., in atherapeutically-effective amount.

In one aspect, the disclosure features a method of treating a patienthaving a glucocerebrosidase deficiency that includes administering a GCBcomposition described herein.

In certain embodiments, the glucocerebrosidase deficiency is Gaucherdisease.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepracticing or testing of the present invention, suitable materials andmethods are described below. All cited publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

Other features, objects, and advantages of the invention will beapparent from the description and from the claims.

DETAILED DESCRIPTION Overview

Compositions of free thiol-containing proteins (e.g., GCB) arerelatively unstable as liquid compositions. The three exposed free thiolgroups in GCB can undergo reactions which lead to reduction instability, e.g., by aggregation of GCB molecules. For example, in bufferat a pH of 6, typically 1-2% of the protein has aggregated upon onemonth of storage and about 15% has aggregated after 6 months of storage.While not wishing to be bound strictly by theory or mechanism, it isbelieved that a number of factors contribute to protein instability,e.g., the aggregation reaction: For example, free O₂ in solution canaccelerate cross-linking of the free thiol groups, leading toaggregation. If the reaction of the free thiol groups is reduced and/orif the protein can be made more compact, for example, by buryingcysteine residues in hydrophobic domains, protein aggregation can bereduced. In addition, protein degradation (e.g., fragmentation) can bereduced.

Embodiments described herein include one or more measures to address oneor more of these issues. As examples, various factors have beenaddressed to increase the stability of compositions (e.g., liquidcompositions) of free thiol-containing proteins, for example: thepresence of reactive species (e.g., free O₂) in solution, theavailability of free sulfhydryl groups (e.g., free thiols) on theprotein, the protein conformation, and pH. One, two, three, four, or allof these factors can be altered or controlled to increase the stabilityof a protein of interest.

Free Thiol-Containing Proteins

Free thiol-bearing proteins are proteins which in active form have oneor more —S—H moieties. In preferred embodiments, the —S—H moiety isaccessible to a reactant and can react with that reactant, e.g., areducing agent such as cysteine, under conditions which are optimal forstability. Alternatively, the —S—H moiety can react with a reactantunder physiological conditions with one or more biological fluids thatit comes into contact with when administered to a patient, e.g., themoiety is accessible for reaction in blood.

A particularly preferred free thiol-containing protein isglucocerebrosidase (GCB). The structure of GCB in solution providesrelatively accessible (as opposed to buried or hindered) free —S—Hmoieties, which promotes reactions with the —S—H moiety.

Another particularly preferred free thiol-containing protein is basicfibroblast growth factor (bFGF).

Other examples of free thiol-containing proteins include: acidicfibroblast growth factor (aFGF), hemoglobin, thioredoxin, calcium- andintegrin-binding protein 1 (CIB1), beta-lactoglobulin B,beta-lactoglobulin AB, serum albumin, antibodies (e.g., humanantibodies, e.g., IgA (e.g., dimeric IgA), IgG (e.g., IgG2), and IgM;recombinant human antibodies), antibody fragments (e.g., Fab′ fragments,F(ab′)₂ fragments, single-chain Fv fragments (scFv)), antibodies andantibody fragments (e.g., Fab′, e.g., monoclonal antibody fragmentC46.3; and scFv) that have been engineered (e.g., so that the antibodyor antibody fragment can be labeled, e.g., with 99mTc, for clinicalimaging) to introduce cysteine residues (e.g., in the third heavy chainconstant domain, e.g., at position 442 in EU/OU numbering; monoclonalantibody MN-14 (a high-affinity anti-carcinoembryonic antigen(CEA)mab)), core 2 beta 1,6-N-acetylglucosaminyltransferase-M (C2GnT-M),core 2 beta 1,6-N-acetylglucosaminyltransferase-I (C2GnT-I),platelet-derived growth factor receptor-beta (PDGF-beta), adeninenucleotide translocase (ANT), p53 tumor suppressor protein, glutenproteins, acid sphingomyelinase (recombinant acid sphyngomyelinase),desfuroylceftiofur (DFC), apolipoprotein B100 (apoB) and other lowdensity lipoprotein domains, apolipoprotein A-I variants (e.g.,apolipoprotein A-I (Milano) and apolipoprotein A-I (Paris)),hypoxia-inducible factor-1 alpha (HIF-1 alpha), von Willebrand factor(VWF), proteins and peptide mimetics that contain the CAAX motif (e.g.,Ras), mucolytics, carboxypeptidase Y, cathepsin B, cathepsin C, skeletalmuscle Ca²⁺ release channel/ryanodine receptor (RyR1), nuclear factorkappa B (NF-KB), AP-1, protein-disulfide isomerase (PDI), glycoprotein1b alpha (GP1b alpha), calcineurin (CaN), fibrillin-1, CD4, S100A3 (alsoknown as S100E), ionotropic glutamate receptors, humaninter-alpha-inhibitor heavy chain 1, alpha2-antiplasmin (alpha2AP),thrombospondin (also known as glycoprotein G), gelsolin, mucins,creatine kinase (e.g., S-thiomethyl-modified creatine kinase), FactorVIII, phospholipase D (PLD), insulin receptor beta subunit,acetylcholinesterase, prochymosin, modified alpha 2-macroglobulin (alpha2M) (e.g., proteinase- or methylamine-reacted alpha 2M), glutathionereductase (GR), complement component C2 (e.g., 2a), complement componentC3 (e.g., C3b), complement component 4 (e.g., 4d), complement Factor B(e.g., Bb), alpha-lactalbumin, beta-D-galactosidase, endoplasmicreticulum Ca²⁺-ATPase, RNase inhibitor, lipocortin 1 (also known asannexin 1), proliferating cell nuclear antigen (PCNA), actin (e.g.,globular actin), coenzyme A (CoA), acyl-CoA synthetase (e.g.,butyryl-coenzyme A synthetase), 3-2trans-enoyl-CoA-isomerase precursor,atrial natriuretic factor (ANF)-sensitive guanylate cyclase,Pz-peptidase, aldehyde dehydrogenase (e.g., acylated aldehydedehydrogenase), P-450 and NADPH-P-450 reductase,glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 6-pyruvoyltetrahydropterin synthetase, lutropin receptor, low moleculat weightacid phosphatase, serum cholinesterase (BChE), adrenodoxin,hyaluronidase, carnitine acyltransferases, interleukin-2 (IL-2),phosphoglycerate kinase, insulin-degrading enzyme (IDE), cytochrome c1heme subunit, S-protein, valyl-tRNA synthetase (VRS), alpha-amylase I,muscle AMP deaminase, lactate dehydrogenase, and somatostatin-bindingprotein. Also included are fragments of such proteins (e.g., activedomains, structural domains, dominant negative fragments, and so forth).The proteins containing a free thiol group can be naturally occurringproteins, recombinant proteins, proteins modified (e.g., by recombinantDNA technology) to contain a cysteine residue, or proteins chemically orenzymatically treated so that a sulfhydryl moiety on a cysteine residueis in a reduced state, i.e., to have a free —S—H moiety.

In some embodiments, a composition described herein includes a proteinhaving a naturally occurring sequence. In other embodiments, thesequence of the protein will differ at 1, 2, 3, 4, 5, or up to 10 aminoacid residues from a naturally occurring sequence. In other embodiments,the amino acid sequence of the protein will differ by 1, 2, 3, 4, 5, orup to 10% from a naturally occurring sequence.

Reactive Species Removal

Reactive species, e.g., O₂ or peroxides, dissolved in solution candecrease the stability of a protein in the composition, e.g., bypromoting protein aggregation. However, even in the absence of O₂, free—S—H moieties can cross-link.

To increase protein stability, reactive species, e.g., O₂, in a solutioncan be removed, e.g., chemically, by the use of O₂ scavengers, e.g.,sulfites. Chemical scavengers are often less desirable as they can causeprotein degradation. O₂ can also be removed physically from a solution,e.g., by degassing the solution, e.g., by applying a vacuum to thesolution to remove the O₂ from solution and replacing it with an inertgas, e.g., nitrogen or argon. Reduction of O₂ levels can also beaccomplished physically by purging a solution with a gas other than O₂,e.g., an inert gas, e.g., nitrogen or argon. Purging can be accomplishedby bubbling the gas through the solution to be purged of O₂.

With protein (e.g., GCB) compositions, bubbling or other manipulationswhich result in interfaces between a gas and a protein-containingsolution are often avoided in the treatment of proteins because they candenature proteins, however, these manipulations have been discovered tobe well-tolerated in the GCB methods disclosed herein.

O₂ removal can be combined with minimization of contact of a solutionwith O₂, e.g., by manipulation and storage under conditions whichminimize the presence of O₂, e.g., filling of containers under a gasother than O₂, e.g., an inert gas, e.g., nitrogen or argon, or thesealing of containers with such a gas. In general, it is desirable tominimize the contact of the solution with O₂ prior to administration tothe patient. O₂ levels in head space should be reduced to less thanabout 10%, preferably less than about 5%, and more preferably less thanabout 2%.

Removal of reactive species may also result in increased proteinstability, e.g., by minimizing oxidation of other moieties as well,e.g., Tyr, Trp, and/or Met residues. In particular, it is desirable tominimize oxidation of these moieties in GCB.

One can test a candidate method for removal of O₂ by providing acomposition containing 2 mg/ml GCB, 0.075% cysteine (as an antioxidant),16% sucrose (to decrease —S—H availability), adjusting the pH to 5.7,and applying the candidate method. The stability of the GCB compositionproduced by the candidate method, measured, e.g., as a percentaggregation or degradation, at a predetermined time is compared with oneor more standards. For example, a suitable standard would be acomposition similar to the test conditions except that an O₂ removalmethod is not applied. The stabilities of the treated (wherein thecandidate O₂ removal method is applied) and untreated (wherein an O₂removal method is not applied) compositions are compared. Suitabilitycan be shown by the test treatment increasing stability as compared withthis standard. Another standard can be a composition similar to the testcomposition except that in place of the candidate method of removal, O₂is removed by a method described herein, for example, by purging ordegassing with an inert gas. Suitability can be shown by the candidatemethod having comparable or better effects on stability than the methoddescribed herein.

Protein stability can be measured, e.g., by measuring proteinaggregation or protein degradation. Protein aggregation can bedetermined, e.g., by size exclusion chromatography, non-denaturing PAGE,or other methods for determining size, etc. Protein degradation can bedetermined, e.g., by reverse phase HPLC, non-denaturing PAGE,ion-exchange chromatography, peptide mapping, or similar methods.

Antioxidants

The stability of a protein in a composition can be increased (e.g.,cross-linking mediated by free —S—H moieties can be reduced) by theaddition of an antioxidant, and in particular, an anti-oxidant whichincludes a moiety which reacts with the free —S—H (e.g., an —S—H), e.g.,cysteine, cysteine-HCl, or methionine. For a protein (e.g., GCB) thatcontains both free thiol groups and also internal disulfide linkageswithin the protein molecule, the level of antioxidant (e.g., cysteine)used should be high enough to minimize cross-linking of the free thiolbonds (e.g., aggregation) but low enough so as not to causefragmentation, and/or proteolysis, and/or degradation (e.g., detectablewith reverse-phase HPLC). For example, with cysteine, particularly forGCB, inclusion of about 0.001% to about 10%, e.g., about 0.01 to about0.15%, e.g., about 0.05% to about 0.1%, is suitable. Levels over 10% maynot be optimal.

For example, one can test a candidate antioxidant (which can be anyagent that can remove or reduce dissolved O₂ in solution) by providing acomposition containing 2 mg/ml GCB, 16% sucrose (to decrease —S—Havailability), adjusting the pH to 5.7, adding the candidate antioxidant(e.g., in an amount described herein, e.g., 0.075%), and purging thecomposition of O₂. The stability of the GCB composition containing thecandidate antioxidant, measured, e.g., as a percent aggregation ordegradation, at a predetermined time is compared with one or morestandards. For example, a suitable standard would be a compositionsimilar to the test conditions except that an antioxidant is not addedto the composition. The stabilities of the treated (containing theantioxidant) and untreated (lacking an antioxidant) compositions arecompared. Suitability can be shown by the test treatment increasingstability as compared with this standard. Another standard can be acomposition similar to the test composition except that in place of thecandidate antioxidant, an antioxidant described herein, for example,cysteine (e.g., in an amount described herein, e.g., 0.075%), is addedto the composition. Suitability can be shown by the candidateantioxidant having comparable or better effects on stability than anantioxidant described herein. If the candidate antioxidant is determinedto be suitable (e.g., it increases stability of the composition ascompared to one of the standards), the concentration of the candidateantioxidant can be refined. For example, the concentration can beincreased or decreased over a range of values and compared to thestandard and to the other concentrations being tested to determine whichconcentration causes the greatest increase in stability.

Protein stability can be measured, e.g., by measuring proteinaggregation or protein degradation. Protein aggregation can bedetermined, e.g., by size exclusion chromatography, non-denaturing PAGE,or other methods for determining size, etc. Protein degradation can bedetermined, e.g., by reverse phase HPLC, non-denaturing PAGE,ion-exchange chromatography, peptide mapping, or similar methods.

A preferred antioxidant is cysteine. Other antioxidants suitable for useinclude: cysteine-HCl, reduced glutathione, thioethanolamine,thiodiglycol, thioacetic acid, monothioglycerol, N-acetylcysteine,dithiothreitol, DL-thioctic acid, mercaptoethanol, dimercaptopropanol,bisulfite, dihydroacorbate, metabisulfite, sulfite, formaldehydesulfoxylate, thiosulfate, and acetone bisulfite. In some embodiments, acombination of two or more of these antioxidants is used in thecompositions described herein. The suitability of the combination can betested as described above for a candidate antioxidant.

Addition of anti-oxidants can result in increased protein stability,e.g., by minimizing oxidation of other moieties as well, e.g., Tyr, Trp,and/or Met residues. In particular, it is desirable to minimizeoxidation of these moieties in GCB.

Carbohydrates

In some embodiments, a carbohydrate is included in the composition.E.g., a carbohydrate can cause the protein to be more compact, and forexample, bury or otherwise hinder access to a moiety, e.g., a cysteineresidue (e.g., a free —S—H moiety on a cysteine residue), e.g., acysteine residue in a hydrophobic domain. This can (e.g., with GCB)increase protein stability, e.g., by reducing protein aggregation.

Carbohydrates include non-reducing sugars, e.g., non-reducingdisaccharides, e.g., sucrose or trehalose, which are suitable for thispurpose. The level of sugar in the composition can be critical. A sugarcontent of about 1 to about 40%, e.g., about 5 to about 30%, e.g., about8 to about 24%, e.g., about 16%, weight per volume (w/v) is suitable,e.g., for use with GCB. A sugar content of about 3 to about 5% is alsosuitable.

One can test a candidate substance, e.g., a candidate carbohydrate, fordecreasing —S—H availability by providing a composition containing 2mg/ml GCB, 0.075% cysteine (as an antioxidant), adjusting the pH to 5.7,adding the candidate substance (e.g., in an amount described herein,e.g., 16%), and purging the composition of O₂. The stability of the GCBcomposition containing the candidate substance, measured, e.g., as apercent aggregation or degradation, at a predetermined time is comparedwith one or more standards. For example, a suitable standard would be acomposition similar to the test conditions except that a substance isnot added to the composition. The stabilities of the treated (containingthe substance) and untreated (lacking a substance) compositions arecompared. Suitability can be shown by the test treatment increasingstability as compared with this standard. Another standard can be acomposition similar to the test composition except that in place of thecandidate substance, a substance described herein, for example, sucrose(e.g., in an amount described herein, e.g., 16%), is added to thecomposition. Suitability can be shown by the candidate substance havingcomparable or better effects on stability than a substance describedherein. If the candidate substance is determined to be suitable (e.g.,it increases stability of the composition as compared to one of thestandards), the concentration of the candidate substance can be refined.For example, the concentration can be increased or decreased over arange of values and compared to the standard and to the otherconcentrations being tested to determine which concentration causes thegreatest increase in stability.

Protein stability can be measured, e.g., by measuring proteinaggregation or protein degradation. Protein aggregation can bedetermined, e.g., by size exclusion chromatography, non-denaturing PAGE,or other methods for determining size, etc. Protein degradation can bedetermined, e.g., by reverse phase HPLC, non-denaturing PAGE,ion-exchange chromatography, peptide mapping, or similar methods.

Preferred carbohydrates are trehalose or sucrose. Other preferredsubstances suitable for use include: maltose, raffinose, glucose,sorbitol. Other suitable substances that can be used to stabilize theprotein include: carbohydrates such as lactose and arabinose; polyolssuch as mannitol, glycerol, and xylitol; amino acids such as glycine,arginine, lysine, histidine, alanine, methionine, and leucine; andpolymers such as PEG, poloxomers, dextran, polypropylene glycol,polysaccharides, methylcellulose, sodium carboxymethyl cellulose,polyvinyl pyrrolidone (PVP), hydrolyzed gelatin, and human albumin. Insome embodiments, a combination of two or more of these carbohydrates(e.g., sucrose and trehalose) is used in the compositions describedherein. The suitability of the combination can be tested as describedabove for a candidate carbohydrate.

pH

pH can be critical in achieving an optimized protein composition, e.g.,a liquid protein composition with increased stability. pH can work byaffecting the conformation and/or aggregation and/or degradation and/orthe reactivity of the protein. For example, at a higher pH, O₂ can bemore reactive. The pH is preferably less than 7.0, more preferably inthe range of about 4.5 to about 6.5, more preferably about 5.0 to about6.0, and more preferably about 5.5 to about 5.8, more preferably about5.7. With some proteins, e.g., GCB, aggregation can reach undesirablelevels at a pH above 7.0 and degradation (e.g., fragmentation) can reachundesirable levels at a pH under 4.5 or 5.0, or at a pH above 6.5 or7.0.

One can test a candidate pH by providing a composition containing 2mg/ml GCB, 0.075% cysteine (as an antioxidant), 16% sucrose (to decrease—S—H availability), adjusting the composition to a candidate pH, andpurging the composition of O₂. The stability of the GCB composition atthe candidate pH, measured, e.g., as a percent aggregation ordegradation, at a predetermined time is compared with one or morestandards. For example, a suitable standard would be a compositionsimilar to the test conditions except that the pH of the composition isnot adjusted. The stabilities of the treated (the composition adjustedto the candidate pH) and untreated (the pH is not adjusted) compositionsare compared. Suitability can be shown by the test treatment increasingstability as compared with this standard. Another standard can be acomposition similar to the test composition except that in place of thecandidate pH, the composition has a pH described herein, for example, pH5.7. Suitability can be shown by the composition at the candidate pHhaving comparable or better effects on stability than the composition atpH 5.7.

Protein stability can be measured, e.g., by measuring proteinaggregation or protein degradation. Protein aggregation can bedetermined, e.g., by size exclusion chromatography, non-denaturing PAGE,or other methods for determining size, etc. Protein degradation can bedetermined, e.g., by reverse phase HPLC, non-denaturing PAGE,ion-exchange chromatography, peptide mapping, or similar methods.

Buffers that can be used to adjust the pH of a protein compositioninclude: histidine, citrate, phosphate, glycine, succinate, acetate,glutamate, Tris, tartrate, aspartate, maleate, and lactate. A preferredbuffer is citrate.

Protein Concentration

A preferred protein (e.g., GCB) concentration can be between about 0.1to about 40 mg/ml, more preferably about 0.5 to about 10 mg/ml, e.g.,about 2 to about 8 mg/ml or about 5 mg/ml.

One can test for a suitable protein concentration by providing acomposition containing 0.075% cysteine (as an antioxidant), 16% sucrose(to decrease —S—H availability), adjusting the pH to 5.7, adjusting theprotein (e.g., GCB) to a candidate concentration, and purging thecomposition of O₂. The stability of the protein (e.g., GCB) compositionat the candidate concentration, measured, e.g., as a percent aggregationor degradation, at a predetermined time is compared with one or morestandards. For example, a suitable standard would be a compositionsimilar to the test conditions except that the protein (e.g., GCB)concentration is a concentration described herein, e.g., 2 mg/ml. Thestabilities of the protein (e.g., GCB) at each concentration arecompared. Suitability can be shown by the candidate concentration havingcomparable or better effects on stability than a concentration describedherein.

Protein stability can be measured, e.g., by measuring proteinaggregation or protein degradation. Protein aggregation can bedetermined, e.g., by size exclusion chromatography, non-denaturing PAGE,or other methods for determining size, etc. Protein degradation can bedetermined, e.g., by reverse phase HPLC, non-denaturing PAGE,ion-exchange chromatography, peptide mapping, or similar methods.

Surfactants

A surfactant can be added to the liquid protein (e.g., GCB) composition.In a preferred embodiment, this can increase protein stability, e.g.,reduce protein degradation, e.g., due to air/liquid interface uponshaking/shipment. A surfactant that increases protein stability, e.g.,does not cause protein degradation, in the liquid composition isselected. A surfactant suitable for use is e.g., poloxamer 188, e.g.,PLURONIC® F68. The surfactant can be present in an amount between about0.005% and about 5%, e.g., between about 0.01% and about 1%, e.g., about0.025% and about 0.5%, e.g., about 0.03% and about 0.25%, e.g., about0.04 to about 0.1%, e.g., about 0.05% to about 0.075%, e.g., 0.05%.

Ideally, a surfactant selected for use in the protein compositionsdescribed herein is one that is not modified, e.g., cleaved, by theprotein.

For example, one can test a candidate surfactant by providing acomposition containing 2 mg/ml GCB, 0.075% cysteine (as an antioxidant),16% sucrose (to decrease —S—H availability), adjusting the pH to 5.7,adding the candidate surfactant (e.g., in an amount described herein,e.g., 0.05%), and purging the composition of O₂. The stability of theGCB composition containing the candidate surfactant, measured, e.g., asa percent aggregation or degradation, at a predetermined time iscompared with one or more standards. For example, a suitable standardwould be a composition similar to the test conditions except that asurfactant is not added to the composition. The stabilities of thetreated (containing the surfactant) and untreated (lacking a surfactant)compositions are compared in conditions simulating “real world”scenarios, e.g., shipping. Suitability can be shown by the testtreatment increasing stability as compared with this standard. Anotherstandard can be a composition similar to the test composition exceptthat in place of the candidate surfactant, a surfactant describedherein, for example, poloxamer 188 (e.g., in an amount described herein,e.g., 0.05%), is added to the composition. Suitability can be shown bythe candidate surfactant having comparable or better effects onstability than a surfactant described herein. If the candidatesurfactant is determined to be suitable (e.g., it increases stability ofthe composition as compared to one of the standards), the concentrationof the candidate surfactant can be refined. For example, theconcentration can be increased or decreased over a range of values andcompared to the standard and to the other concentrations being tested todetermine which concentration causes the greatest increase in stability.

In some embodiments, a combination of two or more surfactants is used inthe compositions described herein. The suitability of the combinationcan be tested as described above for a candidate surfactant.

Protein stability can be measured, e.g., by measuring proteinaggregation or protein degradation. Protein aggregation can bedetermined, e.g., by size exclusion chromatography, non-denaturing PAGE,or other methods for determining size, etc. Protein degradation can bedetermined, e.g., by reverse phase HPLC, non-denaturing PAGE,ion-exchange chromatography, peptide mapping, or similar methods.

GCB

Gaucher disease is an autosomal recessive lysosomal storage disordercharacterized by a deficiency in the lysosomal enzyme,glucocerebrosidase (GCB). GCB hydrolyzes the glycolipid glucocerebrosidethat is formed after degradation of glycosphingolipids in the membranesof white blood cells and red blood cells. The deficiency in this enzymecauses glucocerebroside to accumulate in large quantities in thelysosomes of phagocytic cells located in the liver, spleen, and bonemarrow of Gaucher patients. Accumulation of these molecules causes arange of clinical manifestations including splenomegaly, hepatomegaly,skeletal disorder, thrombocytopenia and anemia. (Beutler et al. Gaucherdisease; In: The Metabolic and Molecular Bases of Inherited Disease(McGraw-Hill, Inc, New York, 1995) pp. 2625-2639).

Treatments for patients suffering from this disease includeadministration of analgesics for relief of bone pain, blood and platelettransfusions and, in some cases, splenectomy. Joint replacement issometimes necessary for patients who experience bone erosion. Enzymereplacement therapy with GCB has been used as a treatment for Gaucherdisease.

The structure of GCB in solution provides relatively accessible (asopposed to buried or hindered) free —S—H moieties, which promotesreactions with the —S—H moiety.

GCB can be obtained by methods that are known in the art. For example,WO02/15927, WO2005/089047, WO03/056897, WO01/77307, WO01/07078, andWO90/07573; European Published App. No. EP1392826; U.S. PublishedApplication Nos. 2005-0026249, 2005-0019861, 2002-0168750, 2005-0265988,2004-0043457, 2003-0215435, and 2003-0133924; and U.S. patentapplication Ser. No. 10/968,870; U.S. Pat. Nos. 7,138,262, 6,451,600,6,074,864, 5,879,680, 5,549,892, 5,236,838, and 3,910,822 describemethods or preparing GCB protein. Any of the GCB protein preparationsdescribed in these patents and applications can be formulated into acomposition described herein.

GCB enzymatic activity can be measured as described in the examplesprovided herein, or as described in the art, e.g., in U.S. Pat. No.7,138,262.

Packaging and Delivery

Protein compositions, e.g., GCB compositions, e.g., the compositionsdescribed herein and in WO02/15927, U.S. Published Application Nos.2005-0026249, 2005-0019861, and 2002-0168750, and U.S. patentapplication Ser. Nos. 09/641,471 and 10/968,870, can be packaged in atwo chamber syringe. For example, the composition in lyophilized formcan be placed into a first syringe chamber and a liquid can be presentin a second syringe chamber (see e.g., U.S. Published Application No.2004-0249339).

Protein compositions, e.g., GCB compositions, e.g., the compositionsdescribed herein and in WO02/15927, U.S. Published Application Nos.2005-0026249, 2005-0019861, and 2002-0168750, and U.S. patentapplication Ser. Nos. 09/641,471 and 10/968,870, can be packaged in aneedleless syringe (see e.g., U.S. Pat. Nos. 6,406,455 and 6,939,324).Briefly, as one example, the injection device includes: a gas chambercontaining a gas or a source of gas; a port which can allow for releaseof gas from the gas chamber; a plunger, which upon the release of gasfrom the gas chamber, can cause movement of at least a first piston; afirst piston; a second piston; a first chamber, e.g. a chamber usefulfor drug storage and mixing; a piston housing, in which are disposed thefirst piston, the second piston and the first chamber; a displacementmember which can, independent of the motive power of gas from the gaschamber, cause movement of one or both of the first and second pistons(the displacement member can be the plunger or a separate member); anorifice suitable for needleless injection in communication with thefirst chamber; wherein the first and second piston, are slideablydisposed within the piston housing, and the displacement member, thesource of gas, and the plunger are disposed such that: in a firstposition of the pistons, a second chamber, e.g., a fluid reservoir, isdefined within the piston housing by the first piston, the pistonhousing and the second piston, the displacement member can move one orboth of the pistons into a second position wherein the first piston isin a position such that the second chamber, which can be a fluidreservoir, is in communication with the first chamber, which can be adrug storage and mixing chamber, and the second piston is moved in thedirection of the first piston, thereby decreasing the volume of thesecond chamber and allowing the transfer of fluid from the secondchamber to the first chamber, the plunger, upon release of gas from thegas chamber, causes the first piston to move so as to decrease thevolume of the first chamber allowing a substance to be expelled throughthe orifice and from the chamber and, e.g., to a subject.

The needleless syringe can include separate modules for a firstcomponent, e.g., a dry or liquid component, and a second component,e.g., a liquid component. The modules can be provided as two separatecomponents and assembled, e.g., by the subject who will administer thecomponent to himself or herself, or by another person, e.g., by anindividual who provides or delivers health care. Together, the modulescan form all or part of the piston housing of devices described herein.The devices can be used to provide any first and second component whereit is desirable to store or provide the components separately andcombine them prior to administration to a subject.

Protein (e.g., GCB) compositions described herein can be incorporatedinto pharmaceutical compositions suitable for administration to asubject, e.g., a human. A GCB composition can include a sufficientdosage of GCB to treat a subject having Gaucher disease. Thepharmaceutical compositions can include one or more pharmaceuticallyacceptable carriers. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,excipients, dispersion media, coatings, antibacterial and antifungalagents, isotonic and adsorption delaying agents, and the like,compatible with pharmaceutical administration. Pharmaceuticalformulation is a well-established art, and is further described, e.g.,in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20thed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); Ansel etal., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed.,Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); andKibbe (ed.), Handbook of Pharmaceutical Excipients AmericanPharmaceutical Association, 3rd ed. (2000) (ISBN: 091733096X). Exceptinsofar as any conventional media or agent is incompatible with theactive compound, such media can be used in the compositions of theinvention. Supplementary active compounds can also be incorporated intothe compositions.

A pharmaceutical composition may include a “therapeutically effectiveamount” of a composition described herein. Such effective amounts can bedetermined based on the effect of the administered composition. Atherapeutically effective amount of a composition may also varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the composition to elicit a desiredresponse in the individual, e.g., amelioration of at least one symptomof a condition or disorder, e.g., a glucocerebrosidase deficiency, e.g.,Gaucher disease. A therapeutically effective amount is also one in whichany toxic or detrimental effects of the composition are outweighed bythe therapeutically beneficial effects.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. For example, thecomposition can be administered by a parenteral mode (e.g., intravenous,subcutaneous, intraperitoneal, or intramuscular injection). The phrases“parenteral administration” and “administered parenterally” as usedherein mean modes of administration other than enteral and topicaladministration, usually by injection, and include, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural, and intrasternal injection and infusion.Preferably, the route of administration is intravenous. Solutions orsuspensions used for parenteral application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders, e.g., lyophilized preparations, for the extemporaneouspreparation of sterile injectable solutions or dispersion. Forintravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyetheyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol, sorbitol, sodium chloride in the composition. Prolongedstability of the injectable compositions can be brought about byincluding in the composition an agent which delays adsorption, forexample, aluminum monostearate, human serum albumin and gelatin.

Sterile injectable solutions can be prepared by incorporating GCBcompositions described herein in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filter sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclewhich contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the composition of sterile injectable solutions, the preferredmethods of composition are vacuum drying and freeze-drying, e.g.,lyophilization, which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The active compounds (e.g., GCB compositions described herein) can beprepared with carriers that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

Protein compositions, e.g., GCB compositions, described herein can beadministered with medical devices known in the art. For example, aprotein (e.g., GCB) composition described herein can be administeredwith a needle-less hypodermic injection device, such as the devicesdisclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413,4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants andmodules useful in the invention include: U.S. Pat. No. 4,487,603, whichdiscloses an implantable micro-infusion pump for dispensing medicationat a controlled rate; U.S. Pat. No. 4,486,194, which discloses atherapeutic device for administering medicants through the skin; U.S.Pat. No. 4,447,233, which discloses a medication infusion pump fordelivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Of course, many other such implants, deliverysystems, and modules also are known.

EXAMPLES Example 1 Materials and Equipment

The following reagents were used in generating the results presented inExamples 2-6:

-   -   GCB: Glucocerebrosidase was prepared using, but not limited to,        the methods described in International App. No. PCT/US01/25882.        Other methods known to one of ordinary skill in the art using        recombinant DNA technology may also be used, for example, the        methods described in International App. Nos. PCT/US88/04314,        PCT/US89/05801, and PCT/US92/00431, and U.S. Pat. Nos.        5,236,838B1, 5,641,670B1, 5,549,892B1, and 6,270,989B1.    -   Sucrose: P/N S-124-01 or S-124-02, Pfanstiehl (Waukegen, Ill.)    -   Cysteine HCl: P/N 2071-05, JTBaker (Phillipsburg, N.J.)    -   Poloxamer 188: P/N P1169, Spectrum (New Brunswick, N.J.)    -   Sodium Citrate: P/N 3649-01, JTBaker (Phillipsburg, N.J.)    -   20 mL vials: P/N 6800-0321, West Pharmaceuticals Services        (Lionville, Pa.)    -   2 mL vials: P/N 6800-0314, West Pharmaceuticals Services        (Lionville, Pa.)    -   20 mm stoppers: P/N 1950-0414, West Pharmaceuticals Services        (Lionville, Pa.)    -   13 mm stoppers: P/N 1950-0412, West Pharmaceuticals Services        (Lionville, Pa.)    -   N₂ gas: P/N UN1066, Airgas (Salem, N.H.)    -   Lyophilizer: Genesis 35EL, SP Industries (Warminster, Pa.)

Example 2 GCB Stability

GCB was formulated at 2.5 mg/mL in 16% sucrose, 0.03% Cysteine HCl,0.05% poloxamer 188, 50 mM sodium citrate, pH 6.0. Twenty-mL glass vialswere filled at 4.5 mL each with the formulated solutions. The filledvials were loaded onto a shelf of a lyophilizer and vacuum degassed at500 mT with a shelf temperature of 20° C. for 3 minutes, followed bybackfill with N₂ to 950 mBar and immediately stoppered with 20 mm graystoppers. The samples were placed into a 2-8° C. stability chamber. At0, 6, 12, 18, and 24 months after storage, the samples were pulled andtested for enzyme activity, aggregation by SE-HPLC, and degradationchanges by RP-HPLC. Enzyme activity was assayed by a colorimetric assayusing p-nitrophenyl β-D-glucopyranoside as the substrate (the activitycan also be assayed, e.g, using the assay described in U.S. Pat. No.7,138,262).

The results are summarized in Table 1. GCB from this composition hadless than 5% changes compared to the baseline after 24 months at 2-8° C.

TABLE 1 Stability Summary for Example 2 after 24 Months Storage at 2-8°C. Time point (months) Activity* SE-HPLC* RP-HPLC* 0 100% 100% 100% 6N/A 100% 100% 12 100% 100% 99% 18  96% 99% 97% 24  95% 99% 96%*Percentage retained from the baseline.

Example 3 Effect of O₂ Levels

GCB was formulated at 2.5 mg/mL GCB in 16% sucrose, 0.03% Cysteine HCl,0.05% poloxamer 188, 50 mM sodium citrate, pH 6.0. Two-mL glass vialswere filled to 1 mL each with the formulated solutions. The headspace ofthe vials was treated using a lyophilizer to have O₂ level of 3%, 6%, or14%. The samples were placed into a 2-8° C. stability chamber. At the 6months time point, the samples were pulled and tested for aggregationchange by SE-HPLC and degradation change by RP-HPLC. The results aresummarized in Table 2. GCB from these compositions is sensitive to theoxygen level in the headspace of the vial. With O₂ less than 3%,essentially no changes were observed after 6 months at 2-8° C.

TABLE 2 Stability Summary for Example 3 after 6 Months Storage at 2-8°C. O2 level in the headspace SE-HPLC* RP-HPLC* 3% 100% 99% 6% 93% 89%14% 64% 72% *Percentage retained from the baseline.

Example 4 Effect of Sucrose Levels

GCB was formulated at 2.5 mg/mL GCB in 0.05% Cysteine HCl, 0.05%poloxamer 188, 50 mM sodium citrate, pH 6.0, containing sucrose levelsof 0%, 5%, 8%, or 16%. Two-mL glass vials were filled to 1 mL each withthe formulated solutions. The vials were vacuum degassed by alyophilizer and overlaid with N₂ to 950 mBar, followed by closing with13 mm stoppers. The samples were placed into a 2-8° C. stabilitychamber. At the 6 month time point, the samples were pulled for testingof aggregation change by SE-HPLC and degradation change by RP-HPLC. Theresults are summarized in Table 3.

TABLE 3 Stability Summary for Example 4 after 6 Months Storage at 2-8°C. Sucrose level SE-HPLC* RP-HPLC* 0% 99.1% 98.9% 5% 99.5% 98.9% 8%99.6% 98.9% 16% 99.8% 98.6% *Percentage retained from the baseline.

Example 5 Effect of Cysteine Levels

GCB was formulated at 2.5 mg/mL GCB in 16% sucrose, 0.05% poloxamer 188,50 mM sodium citrate, pH 6.0, containing cysteine HCL of 0% or 0.05%.Two-mL glass vials were filled to 1 mL each with the formulatedsolutions. The vials were vacuum degassed by a lyophilizer and overlaidwith N₂ to 950 mBar in the headspace, followed by closing with 13 mmstoppers. These samples were placed into a 2-8° C. stability chamber. Atthe 6 month time point, the samples were pulled for testing ofaggregation change by SE-HPLC and degradation change by RP-HPLC. Theresults are summarized in Table 4. Addition of cysteine HCL reduced theaggregation level but increased the degradation level as detected byRP-HPLC.

TABLE 4 Stability Summary for Example 5 after 6 Months Storage at 2-8°C. Cysteine HCL level SE-HPLC* RP-HPLC*   0% 99.4% 99.2% 0.05% 99.8%98.6% *Percentage retained from the baseline.

Example 6 Effect of pH Levels

GCB was formulated at 2.5 mg/mL GCB in 16% sucrose, 0.05% cysteine HCl,0.05% poloxamer 188, 50 mM sodium citrate with pH of 6.0, 5.8 or 5.5.Two-mL glass vials were filled to 1 mL each with the formulatedsolutions. The vials were vacuum degassed and overlaid with N₂ to 950mBar in the headspace, followed by closing with 13 mm stoppers. Thesesamples were placed into a 13-17° C. stability chamber. At the 3 monthtime point, the samples were pulled for testing of aggregation change bySE-HPLC and degradation change by RP-HPLC. The results are summarized inTable 5. Decreasing pH can reduce both the aggregation level and thedegradation level.

TABLE 5 Stability Summary for Example 6 after 3 Months Storage at 13-17°C. pH SE-HPLC* RP-HPLC* 6.0 99.8%  96.9% 5.8 100% 97.9% 5.5 100% 98.5%*Percentage retained from the baseline.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theappended claims. Other aspects, advantages, and modifications are withinthe scope of the following claims.

1.-50. (canceled)
 51. A method of producing a composition comprisingglucocerebrosidase (GCB), the method comprising providing a GCBpreparation and formulating the GCB with a carbohydrate, wherein thecarbohydrate is present in an amount sufficient to maintain thestability of the GCB, wherein the carbohydrate is sucrose or trehalose,and wherein the pH of the composition is less than 7.0.
 52. The methodof claim 51, wherein the stability is at least 5-80% greater, underpre-selected conditions, than the stability of a composition whichdiffers by lacking the carbohydrate.
 53. The method of claim 51, whereinthe carbohydrate is present in an amount sufficient to increase thestability of the GCB.
 54. The method of claim 51, wherein thecarbohydrate is present in an amount sufficient to inhibit the reactionof a free thiol on a first molecule of the GCB with a free thiol on asecond molecule of the GCB to form an aggregate.
 55. The method of claim51, wherein the carbohydrate is present in an amount sufficient toinhibit the formation of an aggregate formed by the reaction of a freethiol on a first molecule of the GCB with a free thiol on a secondmolecule of the GCB by at least 5-80%, under pre-selected conditions, ascompared to the same composition lacking the carbohydrate.
 56. Themethod of claim 51, wherein the carbohydrate is present in an amountsufficient that upon storage, in a gas tight container, at a temperatureof 2-8° C., for a period of 6 months, the composition will retain atleast 85% of the stability the composition had prior to storage.
 57. Themethod of claim 56, wherein the storage occurs in darkness.
 58. Themethod of claim 51, wherein the carbohydrate is present in an amountsufficient to have stability comparable to that of a lyophilizedcomposition comprising sucrose, 0.01% polysorbate-20, pH 6.0, 50 mMCitrate.
 59. The method of claim 51, wherein the carbohydrate is presentin an amount sufficient to maintain biochemical integrity andbioactivity characteristics of the GCB.
 60. The method of claim 51,wherein the pH of the composition is between about 4.5 and about 6.5.61. The method of claim 51, wherein the composition further comprises anantioxidant, wherein the antioxidant and the carbohydrate are present inamounts sufficient to maintain the stability of the GCB.
 62. Thecomposition of claim 61, wherein the antioxidant is cysteine,cysteine-HCl, or methionine.
 63. The method of claim 51, wherein thecomposition further comprises a surfactant.
 64. The method of claim 63,wherein the surfactant is poloxamer
 188. 65. The method of claim 51,wherein the composition contains less than about 10% O₂.
 66. The methodof claim 51, further comprising physical removal of O₂ from thecomposition.
 67. The method of claim 51, wherein the GCB has two, three,or more free thiol groups and has zero, two, four, or more thiol groupswhich form sulfhydryl bridges, per active unit of GCB.
 68. The method ofclaim 51, further comprising exposing the composition to an inert gas,wherein the inert gas is present in a concentration higher than in theambient atmosphere.
 69. The method of claim 51, further comprisingpackaging the composition.
 70. The method of claim 69, wherein thepackaging comprises contacting the GCB with an inert gas to reduce theamount of a reactive species, and introducing the GCB, the inert gas,and the carbohydrate into a gas tight container.
 71. The method of claim70, wherein the container comprises a headspace comprising at least 90%(vol/vol) an inert gas.
 72. The method of claim 71, wherein thecontainer is a prefilled syringe, a vial, or ampoule.
 73. The method ofclaim 72, wherein the prefilled syringe is a needleless syringe.
 74. Themethod of claim 70, wherein the inert gas is N₂ or Ar and the reactivespecies is O₂.
 75. The method of claim 51, wherein the composition is aliquid.
 76. The method of claim 72, wherein carbohydrate is present atbetween about 1 and about 40% (wt/vol).
 77. The method of claim 72,wherein the antioxidant is present at between about 0.001 and about 10%(wt/vol).
 78. The method of claim 72, wherein the composition comprisesabout 0.1-40 mg/ml GCB, about 0.001-10% cysteine, about 1-40% sucrose,at a pH of about 5.5-6.0, and wherein the level of dissolved O₂ is lessthan about 10%.
 79. The method of claim 51, wherein the composition doesnot comprise polysorbate.
 80. A composition produced by the method ofclaim
 51. 81. A method of treating a subject having a glucocerebrosidasedeficiency, the method comprising administering the composition of claim81 to the subject.
 82. The method of claim 81, wherein theglucocerebrosidase deficiency is Gaucher disease.